Predicting hERG repolarization power at 37°C from recordings at room temperature

Mutations in the KCNH2 gene cause long or short QT syndromes (LQTS or SQTS) predisposing to life-threatening arrhythmias. KCNH2 encodes for the voltage-gated K+ channel hERG involved in the late repolarization phase of the cardiac action potential (AP). For the last decades, sequencing KCNH2 has provided a plethora of variants associated or not with clear pathological cardiac phenotypes. Identifying pathogenic or likely pathogenic variants from the benign ones would provide useful information to clarify the genetic background of LQTS patients and relatives, and to stratify the risk of adverse events. In face of a wide spectrum of hERG biophysical defects, we looked for a way to summarize the net loss or gain of function in a unique index. In a previous work, we defined as the repolarization power the time integral of the K+ currents developed during an AP clamp. Here, with the aim of accelerating the functional characterization of hERG variants using automated patch-clamp, we adapted the AP-clamp protocol to establish, at room temperature, at which the recording success rate is high, a repolarization power index, as reliable and informative as the one measured at physiological temperature. We also illustrate that the repolarization power determined at room temperature is predictive of the repolarization power at physiological temperature for 2 pathogenic hERG variants with different biophysical dysfunctions.


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The duration of the QT interval on the surface electrocardiogram (ECG) is a representation of the repolarization time in cardiac ventricles. QT intervals vary as a function of various physiological (age, heart rate, hormones...) or pathophysiological (heart disease, fever, or drug intake) factors 1 . Normal QT intervals, when corrected by heart rate (QTc), are below 470-480 ms. When these QTc intervals are markedly prolonged, generally to values greater than 550-600 ms, polymorphic ventricular tachycardia such as torsade de pointes, elicited by emotions or physical activity, may occur and can degenerate to fatal arrhythmias such as ventricular fibrillation. Excessive QTc prolongations may have various origins: mutations in genes related to ion channels, as in the congenital long QT syndrome (cLQTS) 2 , or upon exposure to stressing environmental conditions as in the acquired long QT syndrome (aLQTS) 3 . aLQTS is frequently provoked by the intake of QT-prolonging drugs, many of them of common use 4 . In this case, reversion back to normal generally follows withdrawal of the causative trigger.
Physiologically, the two voltage-gated K + channels K V 7.1 and hERG, are responsible, as poreforming channel α-subunits, for delayed outward currents, I Ks and I Kr , respectively, involved in the cardiac action potential (AP) repolarization 9 . Their regulation contributes to AP duration adaptation to heart rate. A reduction in outward current and/or an increase in inward current, a condition called reduced repolarization reserve 10 , leads to AP duration lengthening, underlying the QT interval prolongation. About a thousand of cLQTS-associated KCNH2 missense variants have been reported 11 .
Finally, gain-of-function hERG mutants have been detected in patients with abnormally short QT duration (≤ 360 ms) on the ECG, presenting symptoms varying from atrial to ventricular fibrillation and sudden death 12,13 .
For the last decades, sequencing KCNH2 has provided a plethora of variants associated or not with clear pathological cardiac phenotypes 14,15  level. In addition, when functionally investigated, the heterogeneity of the models and approaches prevents from clearly classifying them as illustrated by different studies focusing on the same variants 19-24 . In addition, the maximal hERG current (I hERG ) is often used as single index to quantify the loss or gain of function impact. Such an index may not capture the variation of some biophysical parameters such as a shift of the activation curve or of the inactivation curve because both gates are maximally open when the peak current is measured.
Therefore, in an attempt to standardize the assessment of hERG variants molecular pathogenicity, we have recently designed a hERG phenotyping pipeline 24 . In order to speed up the molecular phenotyping, we designed a new voltage-clamp protocol to determine more than 10 biophysical parameters of hERG current in 35 seconds. To easily summarize the net functional effect of the modified biophysical parameters, we defined a new, simple and unique index, we called repolarization power, to grade the molecular pathogenicity of hERG channels instead of using only the maximal hERG current. This index is the time integral of the current or current density recorded during an AP-shaped voltage-clamp stimulation (AP-clamp), therefore proportional to the total amount of K + ions crossing the membrane during each AP.
The development of the automated patch-clamp allows now to studying up to 384 cells in parallel, boosting the variant functional investigation speed. However, the aforementioned repolarization power, representative of in vivo hERG contribution to repolarization, has to be established at physiological temperature. Indeed, the K + current conducted by hERG channels (I hERG ) depends on temperature but in a complex manner [25][26][27][28] . Recently, Lei and collaborators have developed a short and informative protocol to extensively study hERG behavior and its temperature dependence. Namely, they associated a simple Hodgkin-Huxley kinetic model, an Eyring formulation of the temperature dependence in the model, and a 15-s stimulation protocol (staircase protocol) designed for any patch-clamp set-up, including high-throughput automated systems 29, 30 . All these studies showed that the relative occupancy of the channel open state (total K + conductance) and the rates of activation, deactivation, inactivation, and recovery from inactivation have different temperature sensitivities, the activation being far more temperature sensitive than inactivation 25,26,29,30 . Furthermore, Lei et al. also showed that experimental estimations of temperature coefficients (Q 10 ) are highly protocol dependent. Therefore, estimation of the effects of hERG variants on repolarization in vivo requires one to ideally work at 35-37°C.
On the other hand, as reported by Rajan and colleagues, in CHO, CV1, or HEK cells studied with the Nanion NPC-16 Patchliner Quattro, the patch-clamp success rate, defined in this study as achieving and maintaining a seal resistance above 200 MΩ, at 35°C could be as low as ~15% compared to ~80% at 25 and 15°C 31 . Thus, high temperature represents a limit to obtain Oliveira-Mendes et al. 2023 -Fast determination of hERG repolarization power -Supplementary information 4 high success rates in patch-clamp experiments and will therefore preclude high-throughput evaluation of all hERG variants.
In this report, our aim was to optimize the functional characterization of hERG variants by circumventing these two opposite constraints (physiological temperature and seal resistance in the GΩ range). To do so, we adapted the AP-clamp protocol 24 in such a way that it could be used at room temperature to predict the reference repolarization power of hERG channels at 37°C.
Since hERG gating kinetics are slower at lower temperature, we attempted to mimic, at 10°C lower temperature, the hERG current profile observed at 37°C, by applying slower variation of voltage than the one used for AP-clamp at 37°C. Using AP-shaped voltage stimulations of various time scales applied at different temperatures, we determined that a unique correcting coefficient of 2 can be used to generate a current profile at room temperature that matches the one generated at physiological temperatures. We also illustrate that the repolarization power determined at room temperature is predictive of the repolarization power at physiological temperature for two pathogenic hERG variants with different biophysical dysfunctions.
Ultimately, we plan to use the repolarization power relative to the WT one to classify hERG variants. Large scale variant investigations using the pipeline we designed 24 will help to determine the limits for confidence interval for benign variants.
The cell lines were confirmed to be mycoplasma-free (MycoAlert, Lonza, France).

hERG plasmid transfection
In a first attempt of recording mutant hERG current using the automated patch-clamp system,  34,35 , and the uncompensated liquid junction potential 36 , the stimulation potential was shifted by +32 mV. On the other hand, the holding potential was set to -80 mV, to allow full recovery from inactivation of hERG current (see Figure S2 for intermediary optimization steps, and Figure 1A, the dashed line representing the optimized AP). AP stimulation was repeated 4 times at a frequency of 1 Hz to ensure stability of the parameters.
In order to compensate for reduced hERG channel kinetics at temperatures lower than 37°C, and hence to allow larger current development during AP stimulation, the stimulation protocol Oliveira-Mendes et al. 2023 -Fast determination of hERG repolarization power -Supplementary information 6 was adapted by applying a time factor. For example, when a factor of 2 was used, the AP duration was linearly doubled and the stimulation frequency slowed by 2 (see Figure 1B inset).
Time factors varied from 0.5 to 5. Currents were sampled at 5 to 20 kHz. For each set of experiments, results were obtained with the same cell batch, on different plates (one per temperature), on the same day. Only recordings obtained with seal resistance (R seal ) > 500 MΩ, series resistance (R s ) < 10 MΩ, cell capacitance (C m ) > 10 pF, and with leak current between 0 and -200 pA at -80 mV before the 4 th AP were considered for analysis, achieved with a custom semi-automated R routine. Currents are expressed as currents in pA. Before analyzing the current recordings, the leak current was automatically subtracted at each voltage value, after calculation from its measurement at resting potential, close to the equilibrium potential for K + ions. The repolarization power was calculated as the time integral of the absolute current during the 4 th AP.

Conventional low-throughput electrophysiology
Temperature effects on WT and mutant hERG channels were also investigated on transfected amplifier (all Axon Instruments, Molecular Devices, CA, USA)). Currents were acquired in the whole-cell configuration, filtered at 3 kHz and recorded at a sampling rate of 6.9 to 20 kHz.
Before membrane capacitance and series resistance 70%-compensation, a series of twenty 30-ms steps to -80 mV was applied from a holding potential (HP) of alternatively -70 mV and -90 mV to subsequently off-line calculate C m and R s values from the recorded currents. A 3step protocol was used to test the absence of current rundown/runup (HP =-80 mV, 1 st prepulse: +40 mV during 1 s, 2 nd pre-pulse: -100 mV during 15 ms, test-pulse: +40 mV during 500 ms, every 5 s). Then, an AP was used to clamp the voltage, that derived from the same O'Hara and Rudy model 33 as in automated patch-clamp. Unlike for automated patch-clamp, intracellular fluoride or high external Ca 2+ concentration is not required to easily obtain Gigaseals. Therefore, the AP stimulation was corrected by the calculated liquid junctional potential only (see Figure 2A, the dashed line representing the AP stimulation) 24 . Three repeated APs were enough to stabilize the resulting I hERG current time course. From around 7 20.0°C, Tyrode temperature was gradually raised until it reached 32.0°C in cell bath and APclamp recording was performed every 2.0°C. As in high-throughput experiments, in order to compensate for reduced hERG channel kinetics at lower temperatures, the stimulation protocol was adapted by applying a time factor, from 1 to 3: the same AP stimulation file (Axon text file: *.atf, compatible with Clampex) was used at various frequencies. We kept the typical quality control parameters of conventional patch-clamp to select the cells: R seal > 1GΩ, R s < 10 MΩ at the beginning and the end of the recording, and with less than 200 pA of leak current at -80 mV before the 3 rd AP used to calculate the repolarization power. At the end of the experiment, the pipette potential offset was checked and recordings presenting offset drift > 5 mV were discarded. Currents are expressed as current densities in pA/pF. Before analyzing the current recordings, we subtracted the leak current, calculated at each voltage value from its measurement at resting potential. It has to be mentioned that only cells presenting detectable hERG currents were analyzed in order to be able to compare repolarization powers at various temperatures in the same cell. Data were analyzed and compiled using Clampfit of pClamp 10 suite, Microsoft Excel, and Prism (GraphPad Software, CA, USA). Statistical analyses were processed using Prism with Wilcoxon matched-pair signed rank test, or Mann-Whitney test, when appropriate. Significance level was set to 0.05.

Temperature dramatically affects the success rate in high-throughput automated patch-clamp system
To illustrate the impact of temperature on the success rate of hERG recordings, we first investigated the seal quality quantified with its resistance (R seal ) as a function of temperature using stable cell lines expressing hERG channels. For that purpose, we used the automated patch-clamp system that provides robust information on the temperature effect by dint of great numbers of cells that can be recorded at once. As illustrated in Figure S3A, the percentage of HEK293 cells with high-quality R seal ≥ 1 GΩ decreased significantly when the recording temperature is increased. The success rate dropped from 38 to 13% when measured at 27 and 37°C, respectively. In this case, one could consider that the success rate at 37°C is acceptable although very low. However, if one aims to characterize hundreds of hERG variants, the most realistic approach is to use transient expression which may add another fragilizing factor to seal quality. To test this, we electroporated HEK293 cells with a plasmid over-expressing hERG and estimated again the effect of temperature on seal quality. The percentage of cells reaching a R seal ≥ 1 GΩ at 27°C reached 18% but only 21 cells (5.4% of the plate wells) exhibited hERG currents above 50 pA at +50 mV ( Figure S3C). In contrast, at 37°C, the percentage of cells with R seal ≥ 1 GΩ dropped to 5% and none of them displayed any hERG current. Including cells with R seal < 1 GΩ but ≥ 500 MΩ improved marginally only the Oliveira-Mendes et al. 2023 -Fast determination of hERG repolarization power -Supplementary information 8 rate of cells presenting measurable K + currents (3 cells at 37°C - Figure S3B). On the other hand, for HEK293 cells stably expressing hERG channels, the success rate of 38% at 27°C increased to 77% when the seal resistance limit dropped from 1 GΩ to 500 MΩ. Of note, this seal quality remains largely above previously accepted ones by others 29,31 during automated patch-clamp experiments using stable cell lines (≥ 100 and 200 MΩ, respectively).
However, even the medium-quality experimental conditions with seal resistance values above 500 MΩ are not sufficient to reasonably increase data production yield at physiological temperature if one desires to tackle the biophysical properties of hundreds of hERG variants.
Thus, an alternative protocol is needed to predict at room temperature the in vivo impact of variants. As validation of this new protocol, we compared currents obtained at room and physiological temperatures using automated patch-clamp system for HEK293 cells stably expressing hERG channels and conventional patch-clamp technique for cells transiently overexpressing hERG variants, choosing, in this latter case, gold-standard quality criteria to accurately evaluate the temperature effects.

Limitations
The validation of the correcting value used for a 10°C decrease has been conducted during AP clamp in specific time-and voltage-frames, the readout being the repolarization power preservation. The time factor that we are using is not predictive of the Q 10 of each biophysical parameter but may be regarded as a global correcting value. Further tests would be necessary to challenge the correcting value stability with prolonged AP or in depolarized conditions.
As for the ILT variant of Shaker, another voltage-gated channel, some missense mutations may alter the channel sensitivity to temperature 37 . This would prevent to predict the repolarization power, just by applying the time correcting value of 2. However, such an alteration, due to uncoupling of the voltage sensor from the activation gate is also associated with dramatic changes in the activation voltage-dependence of ILT Shaker 38 . From this observation, we can hypothesize that a hERG variant exhibiting a similar prominent uncoupling between the voltage sensor and the activation gate, would barely activate during the action potential whatever the experimental temperature.
Milnes et al. proposed AP clamp on hERG at physiological temperature to be part of safety testing of novel drug candidates 39 allowing to integrate the drug dissociation/reassociation kinetics in the same voltage protocol. It may be then of interest to use the repolarization index instead of the current amplitude as readout for a more 'physiological IC 50 value determination.
However, if the correcting value of 2 may compensate for the temperature effect on hERG channel behavior, it may not be adapted to binding and unbinding kinetics changes.  Ca 2+ on the surface charge of the membrane and compensation of the liquid junction potential, the higher overshoot allowed development of a much larger K + current (all recordings at 37°C, same cell as A). In this example, the holding potential (HP) was still set at -100 mV and a transient inward current due to hERG deactivation was recorded at the end of the AP repolarization. Therefore, HP of the final AP stimulation protocol was set to -80 mV, limiting the inward K + current but still allowing full recovery of hERG current.